Myosin is found in all eukaryotic cells and appears to be involved in diverse cellular motile processes such as cytokinesis. This laboratory has isolated two different CDNAS for nonmuscle myosin heavy chains (MHCs), MHC-A and -B, which are encoded by two different genes in both human and chickens. We have demonstrated the tissue and cell type- dependent expression for the two MHC MRNAS as well as changes in MRNA expression associated with cell growth and differentiation. Additional neuronal-specific forms of MHC-B, which may be generated by alternative splicing of RNA, have also been demonstrated. To understand the mechanisms responsible for regulating the expression of the nonmuscle MHC gene, we have isolated the genomic clones which encode the promoter and flanking region for human nonmuscle MHC-A. The sequence of the promoter region showed many features typical of a housekeeping gene; there is no TATA element and no functional CAAT box. The GC content is high (average 77%) in the 100 bp immediately both upstream and downstream to transcriptional start sites. Multiple transcriptional start sites are observed. Promoter activity has been monitored using luciferase as a reporter and NIH 3T3 cell as a host cell. The analysis of the 5' deletion mutants of the promoter region demonstrates an essential sequence for the core promoter activity resides in the 36 bp region located from -78 to -113 upstream to the main transcriptional start site. This region includes a single potential AP-2 binding site and a single potential SP1 binding site. Mutation of AP-2 binding site resulted in a 65% loss of promoter activity, indicating the functional importance of this site. We have also begun to screen cosmid human genomic DNA libraries using the 5' end CDNA fragment as well as the CDNA fragments which encode neuronal-specific spliced forms, to obtain the human nonmuscle MHC-B gene. Two overlapping clones were isolated and partially characterized by restriction mapping and sequencing. The clones spanned approximately 55 kbp of genomic DNA which includes part of the 5' untranslated region. Identification of the promoter region of this gene is in progress.